We have used DNA-mediated gene transfer to study gene expression and recombination in in the cells of higher eukaryotes. In one series of experiments the thymidine kinase gene of herpes simplex virus I (tk) and pBR322 were introduced in mouse Ltk-cells by the calcium phosphate method. We have subsequently cloned, sequenced and characterized seven junctions between the foreign DNA and the host genome. In all cases the insertions have occurred into the repetitive components of the mouse genome. We have extended our investigations beyond the study of nonhomologous recombination mentioned above to the study of homologous recombination. In this study we have used a family of plasmids with insertion and deletion mutations in the coding region of tk as substrates for intermolecular recombination events. There were three major findings of these studies. First, we were able to show for the first time that the tk+ phenotype that was generated by the cotransfer of two tk mutants was the result of recombination and not complementation at the protein level. This was accomplished by cloning by the technique of plasmid rescue the sequences responsible for this phenotype. Second, using a novel experimental paradigm we were able to demonstrate that these recombinants were the result of true homologous pairing between the molecules bearing the mutations and not the result of ligations between the molecules bearing the mutations (cut and paste models) or of single-strand annealing between molecules appropriately processed by exonucleases. Third, that there is a sharp discontinuity in the manner in which insertions are handled by the recombination machinery depending on whether they are smaller or larger than 24 base pairs. Most recently, we have developed a cell-free recombination system that utilizes partially purified nuclear extracts from mammalian cells. We are also using this cell-free system to screen extracts from human diploid fibroblasts of patients with diseases such as Bloom's syndrome, ataxia telangiectasia, and xeroderma pigmentosa to determine whether these conditions involve primary or secondary defects in some of the proteins mediating recombination.